Following trauma in the adult central nervous system (CNS) of mammals, injured neurons do not regenerate their transected axons. An important barrier to regeneration is the axon growth inhibitory activity that is present in CNS myelin and that is also associated with the plasma membrane of oligodendrocytes, the cells that synthesize myelin in the CNS (see Schwab M. E., et al., (1993) Ann. Rev. Neurosci. 16, 565–595, for review). The growth inhibitory properties of CNS myelin have been demonstrated in a number of different laboratories by a wide variety of techniques, including plating neurons on myelin substrates or cryostat sections of white matter, and observations of axon contact with mature oligodendrocytes (Schwab, M. E., et al., (1993) Annu. Rev. Neurosci. 16 565–595). Therefore, it is well documented that adult neurons cannot extend neurites over CNS myelin in vitro.
It has also been well documented that removing myelin in vivo improves the success of regenerative growth over the native terrain of the CNS. Regeneration occurs after irradiation of newborn rats, a procedure that kills oligodendrocytes and prevents the appearance of myelin proteins (Savio and Schwab, (1990) Neurobiology 87, 4130–4133). After such a procedure in rats is combined with a corticospinal tract lesion, some corticospinal axons regrow long distances beyond the lesions. Also, in a chick model of spinal cord repair, the onset of myelination correlates with a loss of its regenerative ability of cut axons (Keirstead, et al., (1992) Proc. Nat. Acad. Sci. (USA) 89, 11664–11668). The removal of myelin with anti-galactocerebroside and complement in the embryonic chick spinal cord extends the permissive period for axonal regeneration. These experiments demonstrate a good correlation between myelination and the failure of axons to regenerate in the CNS.
Myelin inhibits axon growth because it contains at least several different growth inhibitory proteins. It has been well documented by us and by others that myelin-associated glycoprotein (MAG) has potent growth inhibitory activity, both in vitro and in vivo (McKerracher, L., et al., (1994) Neuron 13, 805–811; Mukhopadhyay, G., et al., (1994) Neuron 13, 805–811; Li, M., et al., (1996) J. Neurosci. Res. 46, 404–414; Schafer, M., et al., (1996) Neuron 16, 1107–1113). A high molecular weight inhibitory activity has been characterized by Schwab and collaborators, and neutralization of this activity with the IN-1 antibody allows some axons to regenerate in white matter (Schwab, M. E., et al., (1993) Ann. Rev. Neurosci. 16, 565–595; Bregman, B., et al., (1995) Nature 378, 498–501.). We also have evidence that there is an additional growth inhibitory protein in myelin (Xiao, Z., et al., (1997) Soc. Neurosci. Absts. 23, 1994). Clearly, there are multiple inhibitory proteins that stop axon regeneration in mammalian CNS myelin.
In addition to the myelin-derived inhibitors there are also other growth inhibitory molecules expressed in the adult mammalian CNS. Tenacin is a growth inhibitory protein that is expressed in some unmyelinated regions of the CNS (Bartsch, U., et al., (1994) J. Neurosci. 14, 4756–4768) and after lesion tenascin is expressed by astrocytes that border the lesion site (Ajemain and David (1994) J. Comp. Neurol. 340, 233–242). Also growth inhibitory proteins that are proteoglycans are expressed by reactive astrocytes, and these proteins form a barrier to regeneration at the glial scar (McKeon and Silver (1995) Exp. Neurol. 136, 32–43).
While axons damaged in the CNS in vivo do not typically regrow, there have been some reports of long distance axon extension in adult white matter. Such growth has been observed following transplantation of grafted neural tissue (Wictorin, K., et al., (1990) Nature 347, 556–558; Davies, S. J. A., et al., (1994) J. Neurosci. 14, 1596–1612; Isacson, 0. and Deacon, T. W. (1996) Neuroscience 75, 827–837), suggesting that embryonic neurons primed for rapid extension of axons may be less susceptible to growth inhibition. Some embryonic neurons are not susceptible to MAG (Mukhopadhyay, G., et al., (1994) Neuron 13, 805–811), but most embryonic neurons are inhibited by the other myelin inhibitors (Schwab, M. E., et al, (1993) Ann. Rev. Neurosci. 16, 565–595). Therefore, in the cases when axons are able to extend on myelin, signaling through intracellular pathways may play an important role in stimulating, or blocking the inhibition of axon growth. For example, it is known that laminin is able to stimulate rapid neurite growth (Kuhn, T. B., et al., (1995) Neuron 14, 275–285), and we have documented that when laminin is present in sufficient concentration, neurites can extend directly on myelin substrates. These findings suggest the possibility that the stimulation of the integrins, the receptors for laminin, is sufficient to allow axon growth on myelin. Similarly, it has been documented that when the adhesion molecule L1 is expressed ectopically on astrocytes, it can partially overcome their non-permissive substrate properties (Mohajeri, M. H., et al., (1996) Eur. J. Neurosci. 8, 1085–1097). Therefore, neurons can, under appropriate conditions, grow axons on inhibitory substrates, suggesting that the balance of positive to negative growth cues is a critical determinant for the success or failure of axon regrowth after injury.
Growth inhibitory proteins typically cause growth cone collapse, a process that causes dramatic rearrangements to the growth cone cytoskeleton (Bandtlow, C. E., et al., (1993) Science 259, 80–83; Fan, J., et al., (1993) J. Cell Biol. 121 867–878; Li, M., et al., (1996) J. Neurosci. Res. 46, 404–414). One family of proteins that has been implicated in receptor-medicated signaling to the cytoskeleton is the small GTPases of the Rho family (Hall, A. (1996) Ann. Rev. Cell Biol. 10, 31–54). In non-neuronal cells it has been clearly documented that mutations in Rho family members that include Rho, Rac and cdc42, affect adhesion, actin polymerization, and the formation of lamellipodia and filopodia, which are all processes important to motility (Nobes, C. D. and Hall, A. R. (1995) Cell 81, 53–62). There is now good evidence that members of the Rho family regulate axon outgrowth in development. Mutations in Rho-related family members block the extension of axons in Drosophila (Luo, L., et al., (1994) Genes Dev. 8, 1787–1802) and disrupt axonal pathfinding in C. elegans (Zipkin, I. L., et al., (1997) Cell 90, 883–894). More recently it has been shown that the guidance molecule collapsin acts through a Rac-dependent mechanism (Jin, Z. and Strittmatter, S. M. (1997) J. Neurosci. 17, 6256–6263). In transgenic mice that express constitutively active Rac in Purkinje cells, there are alterations in the development of axon terminals and dendritic arborizations (Luo, L., et al., (1996) Nature 379, 837–840). Consistent with the observations in vivo, it was found that dominant negative Rac expressed in PC12 cells disrupts neurite outgrowth in response to NGF (Hutchens, J. A., et al., (1997) Molec. Biol. Cell 8, 481–500). Also, treatment of PC 12 cells with lysophosphatidic acid, a mitogenic phospholipid, causes neurite retraction that is mediated by Rho (Tigyi, G., et al., (1996) J. Neurochem. 66, 537–548). Therefore, different members of the Rho family can exert distinct effects on neurite growth, and in PC 12 cells the activation of Rho is correlated with growth cone collapse. In non-neuronal cells, Rho participates in integrin-dependent signaling (Laudanna, C., et al., (1996) Science 271, 981–983; Udagawa, T. and McIntyre, B. W. (1996) J. Biol. Chem. 271, 12542–12548). The possibility that Rho might play a role within the myelin-derived growth inhibitory system has been studied (Jin, Z. and Strittmatter, S.M. (1997) J. Neurosci. 17, 6256–6263). It was concluded, however, that the inhibitory effects of myelin are not mediated by Rho family members.
A need remains for a means of inactivating the multiple inhibitory proteins present in myelin that prevent axonal regrowth after injury in the CNS.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.